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Abstract

To evaluate the prognostic criteria for identifying cows at an increased risk of a fatal
outcome from acute Escherichia coli mastitis, the potential cut-off
values for five diagnostic parameters associated with a high mortality were determined by
receiver operator characteristic curve analysis. These criteria were hematocrit value
>32%, blood non-esterified fatty acid concentration >0.4 mEq/l,
antithrombin activity <120%, platelet count <15 × 104/ml
and presence of dysstasia. Exceeding the cut-off values for at least three parameters on
day 2 after onset predicted fatality (predictive value 87.5). When these prognostic
criteria were applied to 34 clinical cases, cows that met three criteria were seven times
more likely to die than cows that met fewer than three criteria.

To our knowledge, there are few reports on prediction of fatal acute E. coli
mastitis [7, 15].
Significantly decreased milk production and high bacterial growth in the infected quarters are
reported to predict fatal outcomes following experimental E. coli mammary
infection [7]. Severe systemic signs include elevated
rectal temperature, degree of enophthalmos, rumen contraction rate and signs of depression,
which are reported to predict 48% of fatal outcomes resulting from clinical cases of coliform
mastitis [15].

In our previous study, dysstasia associated with decreased antithrombin activity and platelet
counts along with increased hematocrit (HCT) and blood non-esterified fatty acid (NEFA)
concentrations were confirmed as prognostic parameters associated with a high mortality after
therapeutic treatment of dairy cows with acute E. coli mastitis [6]. The ability of these five parameters to predict fatal
outcomes following acute E. coli mastitis in dairy cows remains to be
validated.

The aim of this study was to evaluate the predictors of fatal outcomes resulting from acute
E. coli mastitis. In our present study, receiver operator characteristic
(ROC) curve analysis was performed to identify potential cut-off values for the five
parameters: dysstasia, antithrombin activity, platelet counts, HCT and NEFA.

Predictors of fatal outcomes resulting from acute E. coli mastitis were
determined by ROC curve analysis of 24 Holstein dairy cows, which were fed in 17 dairy farms
of Hokkaido, Japan. All 24 cows had an E. coli infection that was confirmed
by the identification of the causative pathogen from infected quarter milk samples and one or
more of the following findings on day 1: rectal temperature >40°C, heart rate >120
beats/min, respiratory frequency >30 breaths/min and blood total leukocyte counts
<5,000/µl.

The cut-off values for differentiating between non-survivors and survivors amongst the 24
clinical cases were determined using data on dysstasia, HCT values, NEFA concentration,
antithrombin activity and platelet counts. The sensitivity (proportion of non-survivors that
were predicted to have a fatal outcome), specificity (proportion of survivors that were
predicted to survive), predictive value (proportion of cows predicted to have a fatal outcome
that were indeed non-survivors) and likelihood ratio (sensitivity/[1-specificity]) for
non-survivors were determined for several ranges of the blood test parameters and dysstasia.
ROC curves [sensitivity vs (1-specificity)] were constructed to identify the optimum threshold
among the significant cut-off values. Odds ratios and 95% confidence intervals were determined
to establish significant differences. A P-value of <0.05 was considered
statistically significant.

The utility of the predictors was verified through their use in a trial on day 2 involving 34
clinical cases that were sampled similarly from 32 dairy farms in Hokkaido, Japan. The 34 cows
had E. coli infection that had been confirmed by isolation of bacteria from
infected quarter milk samples and one or more of the following findings on day 1: rectal
temperature >40°C, heart rate >120 beats/min and respiratory frequency >30
breaths/min. All of the tested bacterial isolates were sensitive in vitro to
kanamycin as measured by antimicrobial susceptibility test using the disk diffusion
method.

As the systemic antimicrobial therapy, kanamycin sulfate was administered to cows with acute
E. coli mastitis on days 1, 2 and 3 after the onset. All cows were
administered kanamycin sulfate (4,000–6,000 mg/cow/day) intramuscularly, 7.2% sodium chloride
solution (2,000 ml/cow/day) intravenously, and a combination of kanamycin
sulfate (300 mg/cow/day) and penicillin-G-procaine (300,000 U/cow/day) as an intra-mammary
infusion. Additionally, 1,000 U of heparin sodium (25–50 ml/cow/day),
physiological saline solution (2,000–8,000 ml/cow/day) and 5% glucose
(2,000–5,000 ml/cow/day) were intravenously administered when they were
judged to be necessary. The frequency and dosages of medical treatments were identical for
both survivors and non-survivors. On day 1, quarter milk [8] from affected cows was collected aseptically into sterilized culture tubes.
E. coli isolates were identified using a specific biochemical system
(Sysmex-bioMerieux Co., Ltd., Tokyo, Japan) and a laboratory-designed kit (VITEK 2;
Sysmex-bioMerieux Co., Ltd.) in a diagnostic laboratory (Kishimoto Medical Lab., Tomakomai,
Japan). On day 2, 8–10 ml of peripheral blood was collected from the jugular
vein into evacuated tubes (BD Vacutainer SST II K2 EDTA; Becton, Dickinson and Co., Tokyo,
Japan) and into tubes containing 3.8% sodium citrate as an anticoagulant (NP-CS0457; Nipro,
Tokyo, Japan). Analyses of the four serum parameters were conducted in a diagnostic laboratory
(Kishimoto Medical Lab.) only on day 2 and not on day 1 or 3. Antithrombin activity was
measured by a chromogenic assay (N-Assay L AT III; Nittobo, Tokyo, Japan) with an automated
instrument (JCA-BM12; JEOL, Tokyo, Japan) using plasma treated with sodium citrate. Serum NEFA
concentration was measured using an autoanalyzer (JCA-BM2250; JEOL). HCT, platelet counts and
white blood cell counts were measured with an automated instrument (Analyzer SE-9000; Sysmex)
using plasma treated with EDTA.

The sensitivity, specificity, predictive value and likelihood ratio for non-survivors were
also determined for the 34 clinical cases. Odds ratio and 95% confidence interval around the
odds ratio were determined to establish significant differences.

Cows with acute E. coli mastitis that met the prognostic criteria on day 2,
comprising HCT >32%, NEFA >0.4 mEq/l, antithrombin activity <120%,
platelet counts <15 × 104/ml and presence of dysstasia, were at
higher risk of dying or being euthanized. If at least three of these parameters were met, a
fatal outcome was predicted. From among the 24 clinical cases, cows with three or more
parameters that exceeded the cut-off values were at a significantly
(P<0.01) higher risk of death or euthanasia (Table 1). The fatal outcomes of the affected cows were predicted on day 2
(P<0.01) and day 3 (P<0.01), but not on day 1
(P=0.06). The sensitivity, specificity, predictive value and likelihood
ratio of the prognostic criteria on days 2 and 3 were 100%, 94.1%, 87.5% and 17, and 100%,
83.3%, 75.0% and 6, respectively (Table 1).

When these criteria and cut-off values were applied to 34 other clinical cases, cows with
three or more of the parameters that met the criteria on day 2 were seven times more likely to
die compared with cows with fewer than three of the parameters that met the criteria on day 2
(P<0.05, Table
2). The sensitivity, specificity, predictive value and likelihood
ratio of the prognostic criteria for the cows on day 2 were 62.5%, 80.8%, 50.5% and 3.3,
respectively (Table 2).

When the predictive value of each of these five parameters was considered as an individual
prognostic criterion, dysstasia was the most reliable parameter for diagnosing fatal outcomes
resulting from acute E. coli mastitis. Except for day 1, among these 34 cows,
those with dysstasia were at a significantly higher risk of dying or being euthanized
(P<0.01) (Tables 1 and​and 2 2).

The fatal outcomes resulting from acute E. coli mastitis could be predicted
on day 2 after onset. Cows showing severe clinical signs for three consecutive days after
onset of naturally occurring acute E. coli mastitis were found to be at a
significantly higher risk of dying or being culled [11]. Cows showing severe systemic signs for four consecutive days after experimentally
induced E. coli mammary infections were euthanized [7], whereas cows with no signs on day 2 after infection recovered [7, 10]. Significantly
increased HCT and NEFA concentrations, and significantly decreased antithrombin activity and
platelet counts were observed in non-survivors, compared with survivors on day 2 and day 3,
but not on day 1 [6]. The differences in clinical
symptoms between non-survivors and survivors became apparent starting from day 2 after the
onset of acute E. coli mastitis.

Dysstasia may be a major parameter for predicting fatal outcomes resulting from acute
E. coli mastitis. Recumbency at 2 days after experimental E.
coli mammary infection was reported as a common clinical finding in two cows that
were euthanized because of severe clinical mastitis [7].
Of the 56 cows, a diagnosis of dysstasia at the first clinical examination was made in 12 with
toxic clinical mastitis, with 31 eventually slaughtered or euthanized [1]. Dysstasia was the most reliable predictor of a fatal outcome in this
study (Tables 1 and​and 2). 2). Examinations focused on dysstasia may help to predict fatal
outcomes resulting from acute E. coli mastitis.

This study verified that fatal outcomes for dairy cows with acute E. coli
mastitis can be accurately predicted using specific prognostic criteria and cut-off values of
HCT >32%, NEFA >0.4 mEq/l, antithrombin activity <120% and platelet
counts <15 × 104/ml, along with the presence of dysstasia. The
usefulness of these prognostic criteria and cut-off values was verified in cows with fatal
E. coli mastitis on dairy farms.

Acknowledgments

This study was supported in part by the Okhotsk Agricultural Mutual Aid Association,
Kitami, Hokkaido, Japan.